Glutathione Peroxidase 1
Recombinant DNA Recombinant DNA techniques allow for the alteration of genes, chromosomes, and viruses. These tools have had a significant impact on a wide range of fields; from basic science to industry (1). One common use of recombinant technology is the insertion of a particular gene into an organism that will then express the gene. This will allow the effects of the gene and it's products to be studied. The gene can be unmodified or have particular bases mutated to assess the effects of these modifications. Modifications of genes are a common application of recombinant technology and are very useful in elucidating how a particular gene product may function. Gene products which are commonly study using recombinant techniques include, interfering RNA, enzymes, signaling proteins, and receptor proteins. An example of a protein that has been studied using recombinant technology is Glutathione Peroxidase 1 (GPx-1). Glutathione Peroxidase 1 GPx-1 is a member of the Glutathione Peroxidase family of enzymes. GPx-1 catalyzes the conversion of hydrogen peroxide into water and is the most abundant selenium containing protein in mammals (2). GPx-1 has been shown to play an important role in protecting hemoglobin from being oxidized in the cytosol of red blood cells as well as playing a general role in protection against oxidative stress (3). Other members of the Glutathione Peroxidase family contain cysteine instead of selenocysteine. By using recombinant DNA technology to induce mutations, the question of why is selenocysteine favored in this enzyme when in others cysteine is preferred can be investigated. Expression In order to express GPx-1 it is cloned into a pNIC28-BSA4 expression vector (Figure 1). This vector is a modified pET28 that has a HIS tag and TEV clevage site sequence already incorporated into the vector. This vector is used for bacterial expression. Following bacterial transformation bacteria are grown in the presence of kanamycin. Because pNIC28-BSA4 conveys resistance against kanamycin, bacteria with the vector will survive and those that do not have the vector will die. Expression of the gene is controlled via activation of the lac operon through the use of Isopropyl β-D-1-thiogalactopyranoside (IPTG). Because GPx-1 is a mammalian protein being expressed in bacteria, complications can arise from the differences in codon usage between the species. This is because different species prefer certain codons for a particular amino acid (4). In order to maximize translation the codons found in the human gene for GPx-1 were alter so as to match the codons preferred by E.Coli. The specific cell strain of E.Coli used to express GPx-1 is BL21DE3. This cells are protease deficient. Additionally, these cells express the T7 RNA polymerase which is a very efficient polymerase, meaning more mRNA transcripts. Because there is more mRNA transcripts there should be more protein which is less likely to be degraded due to a lack of proteases. Protein Purification Purification of GPx-1 is a two step procedure. Initially cells are lysed via sonication and loaded onto a nickel column. Because GPx-1 has a HIS tag the protein will bind to the nickel column. GPx-1 is eluted using an imidazole gradient. The collected fractions are tested for the GPx-1 by SDS-PAGE followed by coomassie staining. The fractions containing GPx-1 are then pooled and loaded onto a size exclusion column. The resin of the size exclusion column causes larger proteins to flow through the column faster while smaller proteins will elute slower. This allows impurities to be removed from the GPx-1 containing fractions. Following elution from the size exclusion column, the fractions are tested for GPx-1 via SDS-PAGE. The fractions containing GPx-1 are then pooled and concentrated. Sources 1. Berg P, Mertz JE. Personal reflections on the origins and emergence of recombinant DNA technology. Genetics. 2010; 184: 9-17. 2. Rotruck JT, Hoekstra WG, Pope AL, Ganther HE, Swanson A, and Hafemann D. Relationship of selenium to GSH peroxidase. Fed Proc. 1972; 31: 691-694. 3. Cho CS, Kato GJ, Yang SH, Bae SW, Lee JS, Gladwin MT, Rhee SG. Hydroxyurea-induced expression of glutathione peroxidase 1 in red blood cells of individuals with sickle cell anemia. Antioxidand and Redox Signal. 2010; 13(1): 1-11. 4. Angov E. Codon usage: nature's roadmap to expression and folding of proteins. Biotechnology Journal. 2011; 6(8): 650-659.